Arenaviruses are responsible for significant zoonotic human illnesses and mortality across the globe, inducing Lassa fever in West Africa, lymphocytic choriomeningitis, and numerous regional hemorrhagic fever diseases in South America. These viruses are unable to infect their host cells without the delivery of a large transcriptionally primed replication and gene expression complex, consisting of the viral polymerase (L) directly bound to the nucleoprotein-encapsidated negative-sense genomic RNA (vRNA) segments. Arenavirus L proteins play essential roles in nearly every step of the virus infection cycle, including gene expression, genome replication, and formation of the infectious viral ribonucleoprotein. An accurate mechanistic understanding of L proteins has been hindered by a lack of available biochemical systems and structural models for these multifunctional enzymes. Machupo virus (MACV) is an arenavirus responsible for localized outbreaks of Bolivian hemorrhagic fever. Our lab has pioneered the use of MACV L as a model for arenavirus replication studies, developing multiple experimental systems for the purification and functional analyses of a full-length MACV L. Previous work in our lab has revealed that the catalytic activity of the MACV L protein is completely inhibited due to direct binding by the viral matrix protein (Z). As a result, the L-Z complex is locked in an inactive state on the viral RNA promoter. Moreover, a recently proposed model has suggested that the interaction of L with the 5' termini of the genome segments (5' vRNA) mediates enhanced RNA synthesis activity. The central hypothesis for my proposal is that the RNA synthesis and cap-snatching domains of MACV L are under opposing regulatory influences by the viral matrix protein (Z) and the terminal 5' genomic RNA sequences. The experiments outlined for this project will address the following gaps in our understanding of the MACV system and arenavirus biology: i) the structural mechanisms underlying the regulation of arenavirus L RNA synthesis by its protein and RNA ligands, ii) the biochemical role of the 5' vRNA in modulating L activity, and iii) the catalytic activity and regulation of the MACV L cap-snatching machinery Findings from this project will offer novel insight into the unanswered questions of arenavirus biology, and will certainly be applicable in downstream endeavors for the development of targeted antiviral therapeutic strategies. Our lab has recently made significant progress in the field of polymerase structural biology with the resolved atomic model of the vesicular stomatitis virus (VSV) L protein in complex with its phosphoprotein cofactor. Moreover, our lab has developed the only available in vitro and cell-based systems for the study of MACV L biochemical functions and regulation by its unique cofactors (Z and the 5' vRNA). These experimental systems make MACV the ideal candidate for L structure-function studies. Collectively, these efforts will significantly advance our understanding of arenavirus replication and gene expression, and the regulation of these activities via small viral ligands.